EP1500431A2 - Immobilised affinity medium and its use in separation - Google Patents

Immobilised affinity medium and its use in separation Download PDF

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Publication number
EP1500431A2
EP1500431A2 EP04254214A EP04254214A EP1500431A2 EP 1500431 A2 EP1500431 A2 EP 1500431A2 EP 04254214 A EP04254214 A EP 04254214A EP 04254214 A EP04254214 A EP 04254214A EP 1500431 A2 EP1500431 A2 EP 1500431A2
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EP
European Patent Office
Prior art keywords
support
medium according
medium
abi
density
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EP04254214A
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German (de)
French (fr)
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EP1500431A3 (en
Inventor
Katherine Louise c/o Millipore Angus (U.K.) Ltd.
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Millipore UK Ltd
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Millipore UK Ltd
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Priority claimed from GB0317132A external-priority patent/GB0317132D0/en
Priority claimed from GB0322487A external-priority patent/GB0322487D0/en
Application filed by Millipore UK Ltd filed Critical Millipore UK Ltd
Publication of EP1500431A2 publication Critical patent/EP1500431A2/en
Publication of EP1500431A3 publication Critical patent/EP1500431A3/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
    • B01J20/0211—Compounds of Ti, Zr, Hf
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28042—Shaped bodies; Monolithic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/58—Use in a single column

Definitions

  • This invention relates to a medium and its use in separation.
  • the solid support may be any suitable support known in the art.
  • the support preferably comprises one or more caustic-resistant materials.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Abstract

A medium which comprises a solid support and, attached thereto, one or more ligands selected from ABI and AMBI, wherein the ligand density is from about 30 to about 80 µmole/ml and less than about 1 mmole/g.

Description

    Field of the Invention
  • This invention relates to a medium and its use in separation.
    • Background to the Invention
  • Affinity chromatography may be used for the purification of proteins, for example IgG purification. Affinity chromatography media typically comprise a solid support (e.g. a resin) and, attached thereto, a selection of affinity ligands. The selectivity of the ligands typically depends on a number of factors, for example the pH or ionic strength of the sample undergoing separation. The ionic strength or pH may be varied to enhance selectivity for a particular protein.
  • 2-Aminobenzimidazole (ABI) and aminomethylbenzimidazole (AMBI) are examples of affinity chromatography ligands.
  • Judicious selection of the solid support, ligands and separation conditions allow a reasonable degree of specificity to be attained. However, conventional non-affinity chromatographic processes are typically accompanied by a high degree of non-specific binding. There remains a need for chromatography media having a greater degree of selectivity, particularly towards proteins such as IgG.
    • Summary of the Invention
  • The present invention is based on the discovery that when ABI and/or AMBI are applied at a density of about 30 to about 80 µmole/ml and less than about 1mmole/g (dry weight) to a solid substrate, optimum ligand performance is achieved.
  • A first aspect of the invention is a medium which comprises a solid support and, attached thereto, one or more ligands selected from ABI and AMBI, wherein the ligand density is from about 30 to about 80 µmole/ml and less than about 1 mmole/g.
  • A second aspect of the invention is a method for separating a compound from a sample, the compound capable of binding to ABI and/or AMBI, the method comprising contacting the sample with a medium of the invention.
  • Another aspect of the invention is a method for the manufacture of a medium of the invention, which comprises applying to a solid support, one or more ligands selected from ABI and AMBI.
    • Description of the Invention
  • The term "ABI" as used herein refers to 2-aminobenzimidazole.
  • The term "AMBI" as used herein refers to aminomethylbenzimidazole.
  • A medium of the invention may be obtained by any suitable method known in the art. For example, the medium may be obtained by activating or derivatising the surface of a porous support and then applying one or more ligands to the surface (see, for example, WO-A-98/080603, WO-A-90/09238 and WO-A-90/09237). Optionally, a spacer may be used between the support and a ligand.
  • Typically, a solvent or wetting agent (e.g. water) is used to apply ligands to the support. Rigid media (e.g. silica) hold any wetting agent within their pores. However, soft media (e.g. agarose gel) also expand upon exposure to the wetting agent. For this reason, two measures of ligand densities are used to define the invention: "µmole/ml" and "mmole/g".
  • The normal unit of measure is "µmole/ml". This unit represents the amount of ligand per total expanded volume, and relates directly to the ligand density of the support "in use".
  • To account for the variations between hard and soft media, ligand density is also expressed in units of "mmole/g". This unit of measure is equal to the amount of ligand per unit dry weight of media, thus eliminating the contribution of the wetting agent from the density comparison. For swelled gel media, this dry weight has a significantly different volume than the wetted media.
  • The solid support may be any suitable support known in the art. The support preferably comprises one or more caustic-resistant materials.
  • Caustic is commonly used in the biopharmaceutical industry as a cleanser and/or sanitant. It has been used to clean media between uses, ridding them of any contaminants that might otherwise remain. A caustic solution (used at typical conditions of 0.5M for 1 hour) may be used to inactivate any microbes or viruses that may remain. A problem with most current affinity ligands and some media (such as Protein A on controlled pore glass) is that the ligand as well as the support are often not caustic stable. Consequently, a degradation of performance may occur upon exposure to caustic. Acid rinses are sometimes used instead to avoid degradation, but these are not preferred.
  • Thus, it is preferred that the support is caustic stable. Examples of caustic-resistant materials include agarose, cellulose, zirconia, polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyethersulphone (PES) and polyvinylidene fluoride (PVDF). In a preferred embodiment, the support comprises cross-linked agarose, the proportion of agarose preferably from about 4 to about 8%, more preferably about 6%.
  • The support may be in any suitable form, for example in the form of one or more beads, membranes, non-woven sheets or monolithic structures. The support may be in the form of one or more porous beads.
  • Preferred supports include PVDF membranes (e.g. Durapore®), PES membranes (e.g. Express@), agarose beads, PTFE resins for filters or bead manufacture, non-wovens (e.g. Typar or Tyvec polyethylene) and PTFE membranes. Examples of agarose beads are described in US 4861705, US 5053332 and WO-A-98/58732.
  • Attached to the support are one or more ligands selected from ABI and AMBI. It is preferred that the ligands are present at a density of from about 40 to about 80 µmole/ml and from about 0.5 to about 0.8 mmole/g; more preferably at a density of about 50 µmole/ml and about 0.7 mmole/g.
  • The following Example illustrates the invention.
    • Example
  • The effect of ligand density on the ratio of binding of BSA:IgG was determined for ABI.
  • A number of adsorbents were prepared with varying ligand densities. These were prepared via allyl activation (0.1 ml/ml), bromination (using bromine water) and applying controlled levels of ligand (assuming that a 50% coupling efficiency would be achieved) at room temperature. The ligand densities were determined by titration. The media were then decanted and an aliquot taken to determine the ligand densities using tests described in US 5652348 and US 5945520.
  • Capacity measurements were carried out using 1 ml of adsorbent. The adsorbent was equilibrated using 5 column volumes (CV) of PBS buffer. Human polyclonal IgG or bovine serum albumin (BSA) at 40 mg/ml (2.5 ml) was applied to the column, then washed with 5 CV PBS buffer. Elution was carried out using 5 CV of 0.1 M Glycine/HCl (pH2). The capacity was determined by recording the absorption of the eluent at 280 nm.
  • Figure 1 shows the effect of ligand density on the ratio of binding of BSA:IgG. It is evident that optimum binding occurs when ABI ligands are used at a density of from about 30 to about 80 µmole/ml. In this case, the optimum ligand density is the density at which the ratio of BSA:IgG is lowest but the IgG capacity is sufficiently high for commercial use.

Claims (17)

  1. A medium which comprises a solid support and, attached thereto, one or more ligands selected from ABI and AMBI, wherein the ligand density is from about 30 to about 80 µmole/ml and less than about 1 mmole/g.
  2. A method according to claim 1, wherein the ligand density is from about 40 to about 70 µmole/ml and from about 0.5 to about 0.8 mmole/g.
  3. A medium according to claim 1 or claim 2, wherein the ligand density is about 50 µmole/ml and about 0.7 mmole/g.
  4. A medium according to any preceding claim, wherein the or each ligand is ABI.
  5. A medium according to any preceding claim, wherein the support comprises a caustic-resistant material.
  6. A medium according to claim 5, wherein the support comprises cross-linked agarose.
  7. A medium according to claim 6, wherein the support comprises from about 4 to about 8% agarose.
  8. A medium according to claim 7, wherein the support comprises about 6% agarose.
  9. A medium according to any preceding claim, wherein the support comprises zirconia.
  10. A medium according to any preceding claim, wherein the support comprises polyethylene, polypropylene, polytetrafluoroethylene, polyethersulphone and/or polyvinylidene fluoride.
  11. A medium according to any preceding claim, wherein the support is in the form of one or more beads, membranes, non-woven sheets or monolithic structures.
  12. A medium according to claim 11, wherein the support is in the form of one or more porous beads.
  13. A method for separating a compound from a sample, the compound capable of binding to ABI and/or AMBI, the method comprising contacting the sample with a medium as defined in any preceding claim.
  14. A method according to claim 13, wherein the compound is a protein.
  15. A method according to claim 14, wherein the protein is IgG.
  16. A method for the manufacture of a medium of any of claims 1 to 12, which comprises applying to a solid support, one or more ligands selected from ABI and AMBI at a density of from about 30 to about 80 µmole/ml and less than about 1 mmole/g.
  17. A method according to claim 16, wherein the ligands are applied at a density as defined in claim 2 or claim 3.
EP04254214A 2003-07-22 2004-07-14 Immobilised affinity medium and its use in separation Withdrawn EP1500431A3 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0317132 2003-07-22
GB0317132A GB0317132D0 (en) 2003-07-22 2003-07-22 Medium
GB0322487A GB0322487D0 (en) 2003-09-25 2003-09-25 Medium
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